Until recently, genetically manipulated plants were limited almost exclusively to those events created by application of classical breeding methods. Creation of new plant varieties by breeding was reserved primarily for the most agronomically important crops, such as corn, due to the cost and time needed to identify, cross, and stably fix a gene in the genome, thus creating the desired trait. In comparison, the advent of genetic engineering has resulted in the introduction of many different heterologous genes and subsequent traits into diverse crops including corn, cotton, soybeans, wheat, rice, sunflowers and canola in a more rapid manner. However, the intergression of a new transgene into elite germplasm is still quite a laborious task due to the tissue culturing and back-crossing needed to produce a commercially viable, elite, line.
Several techniques exist which allow for the introduction, plant regeneration, stable integration, and expression of foreign recombinant vectors containing heterologous genes of interest in plant cells. One such technique involves acceleration of microparticles coated with genetic material directly into plant cells (U.S. Pat. No. 4,945,050 to Cornell; U.S. Pat. No. 5,141,131 to DowElanco; and U.S. Pat. No. 5,538,877 and U.S. Pat. No. 5,538,880, both to Dekalb). This technique is commonly referred to as “microparticle bombardment” or “biolistics”. Plants may also be transformed using Agrobacterium technology (U.S. Pat. No. 5,177,010 to University of Toledo, U.S. Pat. No. 5,104,310 to Texas A&M, European Patent Application 0131624B1, European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot, U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, European Patent Applications 116718, 290799, 320500 all to Max Planck, European Patent Applications 604662, 627752 and U.S. Pat. No. 5,591,616 to Japan Tobacco, European Patent Applications 0267159, and 0292435 and U.S. Pat. No. 5,231,019 all to Ciba-Geigy, U.S. Pat. Nos. 5,463,174 and 4,762,785 both to Calgene, and U.S. Pat. Nos. 5,004,863 and 5,159,135 both to Agracetus). Another transformation method involves the use of elongated needle-like microfibers or “whiskers” to transform maize cell suspension cultures (U.S. Pat. Nos. 5,302,523 and 5,464,765 both to Zeneca). In addition, electroporation technology has been used to transform plant cells from which fertile plants have been obtained (WO 87/06614 to Boyce Thompson Institute; U.S. Pat. Nos. 5,472,869 and 5,384,253 both to Dekalb; U.S. Pat. Nos. 5,679,558, 5,641,664, WO9209696 and WO9321335 to Plant Genetic Systems).
Despite all of the technical achievements, genetic transformation and routine production of transgenic plants in a commercially viable, elite, germplasm is still a laborious task. For example, microparticle bombardment, while capable of being used either on individual cells, cell aggregates, or plant tissues, requires preparing DNA-attached gold particles and optimization of an expensive and not yet widely available, “gun” apparatus. Techniques involving Agrobacterium are extremely limited because not all plant species or varieties within a given species are susceptible to infection by the bacterium. Electroporation techniques are not preferred due to the extreme difficulties and cost typically encountered in routinely making protoplast from different plant species and tissues thereof and the concomitant low viability and low transformation rate associated therewith.
Heterologous DNA can be introduced into regenerable plant cell cultures via whiskers-mediated transformation. While a general description of the process can be found in U.S. Pat. Nos. 5,302,523 and 5,464,765, both to Zeneca, no protocols have been published to date for whisker-mediated transformation of embryogenic cotton cultures.
WO 99/38979 describes whisker-mediated transformation of cotton callus, but does not disclose or suggest whisker-mediated transformation of embryogenic cotton suspension cultures.